As the population of western societies on average ages, the number of people affected by bone remodeling-associated diseases such as osteoporosis continues to increase. The development of new therapeutics is hampered by the high failure rates of drug candidates during clinical testing, which is in part due to the poor predictive character of animal models during preclinical drug testing. Co-culture models of osteoblasts and osteoclasts offer an alternative to animal testing and are considered to have the potential to improve drug development processes in the future. However, a robust, scalable, and reproducible 3D model combining osteoblasts and osteoclasts for preclinical drug testing purposes has not been developed to date. Here we review various types of osteoblast–osteoclast co-culture models and outline the remaining obstacles that must be overcome for their successful translation.

NMDA receptors are important players for neuronal differentiation. We previously reported that antagonizing NMDA receptors with APV blocked the growth-promoting effects evoked by the overexpression of specific calcium-permeable or flip-spliced AMPA receptor subunits and of type I transmembrane AMPA receptor regulatory proteins which both exclusively modify apical dendritic length and branching of cortical pyramidal neurons. These findings led us to characterize the role of GluN2B and GluN2A for dendritogenesis using organotypic cultures of rat visual cortex. Antagonizing GluN2B with ifenprodil and Ro25-6981 strongly impaired basal dendritic growth of supra- and infragranular pyramidal cells at DIV 5–10, but no longer at DIV 15–20. Growth recovered after washout, and protein blots revealed an increase of synaptic GluN2B-containing receptors as indicated by a enhanced phosphorylation of the tyrosine 1472 residue. Antagonizing GluN2A with TCN201 and NVP-AAM077 was ineffective at both ages. Dendrite growth of non-pyramidal interneurons was not altered. We attempted to overexpress GluN2A and GluN2B. However, although the constructs delivered currents in HEK cells, there were neither effects on dendrite morphology nor an enhanced sensitivity to NMDA. Further, co-expressing GluN1-1a and GluN2B did not alter dendritic growth. Visualization of overexpressed, tagged GluN2 proteins was successful after immunofluorescence for the tag which delivered rather weak staining in HEK cells as well as in neurons. This suggested that the level of overexpression is too weak to modify dendrite growth. In summary, endogenous GluN2B, but not GluN2A is important for pyramidal cell basal dendritic growth during an early postnatal time window.

To investigate bone formation and resorption in vitro, it is essential to create bone-like microenvironments on cell culture substrates. Here, we present a coating technique to create such a microenvironment on cell culture plastic (CCP) multiwell plates for studying bone remodeling in vitro. Utilizing this coating, we have developed an assay to simultaneously measure cellular mineral formation and resorption in osteoblast and osteoclast coculture models. A composite matrix of collagen type I and carbonated apatitic calcium phosphate was deposited onto CCP in a reproducible manner using a 10× simulated body fluid solution (SBF) supplemented with type I collagen. qPCR analysis and cellular imaging using fluorescence microscopy demonstrated the promotion of osteogenic differentiation, cell attachment, and proliferation of human bone-marrow-derived mesenchymal stem cells on coated substrates. Moreover, human bone-marrow-derived mononuclear cells successfully differentiated into osteoclasts and resorbed the coated substrate. Using the developed coating, an osteoblast and osteoclast coculture system was established, enabling real-time monitoring of mineral formation and resorption. By providing a controlled and physiologically relevant in vitro model, this assay facilitates the screening of therapeutic compounds, the study of bone cell interactions, and the identification of factors influencing bone remodeling, thereby enhancing translational research in bone health.

Osteoclasts (OCs) are important therapeutic targets in the treatment of osteoporosis. The aim of this study was to explore a novel therapeutic approach for osteoporosis using Arcyriaflavin A (ArcyA), a natural compound derived from the marine invertebrate Eudistoma sp. We systematically evaluated the effects of ArcyA on OC differentiation and function in mouse models using molecular biology assays, cellular function analyses and in vivo animal experiments. We also evaluated the efficacy of ArcyA in human cells. The TRAP staining results provide the first clear evidence of the drug’s inhibitory effect, whereby the administration of ArcyA led to a significant reduction in TRAP-positive cells compared to the control group at concentrations that were non-toxic to bone marrow macrophages. Meanwhile, a significant reduction in the number of multinucleated giant cells with more than ten nuclei was observed. Furthermore, similar TRAP staining results were reproduced in human OCs, suggesting that ArcyA has the same effect on OCs derived from human PBMCs. At the molecular level, ArcyA treatment resulted in the downregulation of genes relevant to OC differentiation (NFATc1, cFos and TNFrsf11α), fusion and survival (DCstamp and ATP6v0d2) and resorption function (CTSK, MMP9, integrin β3 and ACP5). A western blot analysis of the corresponding proteins (NFATc1, cFos, CTSK and integrin β3) further confirmed the PCR results. Furthermore, ArcyA-treated OCs produced significantly fewer resorption pits, indicating suppressed bone resorption activity. Consistent with this, in vivo experiments using an ovariectomy (OVX)-induced osteoporosis mouse model showed that ArcyA treatment significantly alleviated bone loss. Mice in the treatment groups had higher BV/TV values, and this therapeutic effect was enhanced in a dose-dependent manner. In addition, our research also showed that IκB could be a potential target for the inhibitory effect of ArcyA. In conclusion, these findings suggest that ArcyA has significant therapeutic potential for the treatment of osteoporosis by inhibiting osteoclastogenesis and bone resorption. Further studies are warranted to explore its clinical applications.